The present invention pertains to polynucleotides derived from Mycobacterium tuberculosis (M. tuberculosis) genes that aid in imparting resistance to antibiotics and chemically related compounds. This invention also relates to the use of the polynucleotides as oligonucleotide primers or probes for detecting M. tuberculosis strains that are resistant to antibiotics and related compounds in a biological sample. The present invention is also directed to diagnostic kits for detecting specific strains of M. tuberculosis expected to be contained in a biological sample.
Tuberculosis remains the world's leading infectious cause of adult deaths due to a single pathogen. Outbreaks of Multidrug-Resistant (MDR) tuberculosis defined as resistant to rifampicin and isoniazid are numerous, with low rates of treatment response and very high mortality. Some of these outbreaks involve patients with HIV infection (1, 2). In some reports, strains with a particular genotype have been identified, such as the “W” strain, which caused a major outbreak in New York (3).
In 1995, it was reported that the largest proportion of the M. tuberculosis strains from the Beijing area had highly similar IS6110 restriction fragment length polymorphism (RFLP) patterns and identical spoligo patterns (4). These strains were therefore designated “Beijing” genotype strains. The “W” variant strains in New York appeared to represent the Beijing genotype of M. tuberculosis. FIG. 1 depicts an example of the characteristic IS6110 RFLP and highly specific spoligo patterns of the Beijing genotype. The unexpected level of genetic conservation among M. tuberculosis isolates of the Beijing genotype led to the hypothesis that these strains may have selective advantages over other M. tuberculosis strains. Moreover, Beijing genotype strains were significantly associated with drug resistance in Cuba, Estonia, and Vietnam (vanSoolingen unpublished).
Several studies have suggested that strains of the Beijing genotype are emerging. In Vietnam, the proportion of Beijing strains was 71% in patients aged under 25, and 41% for those over 55 (vanSoolingen unpublished). Beijing strains have been implicated in several tuberculosis epidemics in the U.S.A. (3) and recently in Gran Canaria (5). A recent study showed that 82% of MDR strains isolated in a prison in Azerbaijan, Eastern Europe, are of the Beijing genotype (6).
Knowledge of a possible molecular basis for the acquisition of drug resistance in M. tuberculosis would be extremely useful as a prerequisite for appropriate treatment. Resistance to antibiotics in M. tuberculosis is due to genomic mutations in specific genes of the bacterium. In contrast to other Gram-positive or Gram-negative pathogens with MDR phenotypes, plasmid or transposon-mediated mechanisms of resistance have not been reported in M. tuberculosis (3,4,5).
Recent studies have provided evidence for a role of mutator phenotypes in the emergence of MDR clinical Pseudomonas isolates (6). Such phenotypes may enable the bacteria, not only to easily acquire resistance to several antibiotics, but also to adapt to new niches and to escape immune surveillance by modulating bacterial resistance to host defenses (7). This prompted an investigation into whether a similar situation might exist in M. tuberculosis. Since MDR strains are dangerous to manipulate, it was decided, as a first step, to simply look for the presence of mutations in genes expected to play a role in mutation frequency.
Mutator phenotypes commonly result from defects in DNA repair enzymes (8). An in silico analysis suggested that most mismatch repair systems, like mutS, mutL or mutH, are missing in the M. tuberculosis genome. However, the frequency of spontaneous mutations in M. tuberculosis (in vitro cultures) is similar to that found in other bacteria carrying mismatch repair systems. This characteristic could have significant implications with respect to genome stability and strain variability (9). This suggests that unknown sequences, similar to genes responsible for the repair of DNA lesions resulting from the alkylation or oxidation of nucleotides, are present in the genome of M. tuberculosis. 
Thus, there was a need in the art to identify sequences in the genome of M. tuberculosis having similarity to genes responsible for the repair of DNA lesions responsible for alkylation or oxidation of nucleotides, such as the mut genes from E. coli. More particularly, there was a need to identify sequences similar to E. coli genes and other mut genes. There was a further need to develop methods of using these sequences to predict the epidemic character of a Mycobacterium tuberculosis isolate and/or a selective advantage to be maintained in the host and/or the acquisition of multiple drug resistance (MDR) by the isolate.